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Published as doi: 10.1096/fj.06-5764fje.
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(The FASEB Journal. 2006;20:2621-2623.)
© 2006 FASEB

The extracellular adherence protein from Staphylococcus aureus abrogates angiogenic responses of endothelial cells by blocking Ras activation

Astrid C. S. Sobke*,1, Dennis Selimovic{dagger}, Valeria Orlova{ddagger},2, Mohamed Hassan#, Triantafyllos Chavakis{ddagger},2, Athanasios N. Athanasopoulos{ddagger}, Uwe Schubert§, Muzaffar Hussain||, Gerald Thiel, Klaus T. Preissner§ and Mathias Herrmann*

* Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, Homburg/Saar, Germany;

{dagger} Clinic of Operative Dentistry and Periodontology, University of Saarland Hospital, Homburg/Saar, Germany;

{ddagger} Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany;

§ Institute of Biochemistry, Justus-Liebig-University, Giessen, Germany,

|| Institute of Medical Microbiology, University Hospital, Münster, Germany,

Department of Medical Biochemistry and Molecular Biology, University of Saarland Hospital, Homburg/Saar, Germany; and

# Department of Dermatology, Heinrich-Heine-University, Düsseldorf, Germany

1Correspondence: Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, D-66421 Homburg/Saar, Germany. E-mail: astrid.sobke{at}gmx.de

ABSTRACT

The extracellular adherence protein (Eap), a broad-spectrum adhesin secreted by Staphylococcus aureus, was previously shown to curb acute inflammatory responses, presumably through its binding to endothelial cell (EC) ICAM-1. Examining the effect of Eap on endothelial function in more detail, we here show that, in addition, Eap functions as a potent angiostatic agent. Concomitant treatment of EC with purified Eap resulted in the complete blockage of the mitogenic and sprouting responses elicited by vascular endothelial growth factor (VEGF)165 or basic fibroblast growth factor (bFGF). Moreover, the induction of tissue factor and decay-accelerating factor were repressed by Eap, as determined by qRT-polymerase chain reaction (qRT-PCR), with a corresponding reduction in Egr-1 protein up-regulation seen. This angiostatic activity was accompanied by a corresponding inhibition in ERK1/2 phosphorylation, while activation of p38 was not affected. Inhibition occurred downstream of tyrosine kinase receptor activation, as comparable effects were seen on TPA-induced ERK1/2 phosphorylation. Similar to previously described angiostatic agents like angiopoietin-1 or the 16-kDa prolactin fragment, Eap blockage of the Ras/Raf/MEK/ERK cascade was localized by pull-down assay at the level of Ras activation. Eap’s combined anti-inflammmatory and antiangiogenic properties render this bacterial protein not only an important virulence factor during S. aureus infection but open new perspectives for therapeutic applications in pathological neovascularization.—Sobke, A. C. S., Selimovic, D., Orlova, V., Hassan, M., Chavakis, T., Athanasopoulos, A. N., Schubert, U., Hussain, M., Thiel, G., Preissner, K. T., Herrmann, M. The extracellular adherence protein from Staphylococcus aureus abrogates angiogenic responses of endothelial cells by blocking Ras activation.


Key Words: proliferation • sprouting • tissue factor • ERK1/2 • Akt/PKB

STAPHYLOCOCCUS AUREUS, a gram-positive bacterium whose natural habitats are the human skin and mucous membranes, is the "classical" and most common causative agent of abscess formation and surgical site infections (SSIs), which today constitute the second most frequent group of nosocomial infections (1 2 3) . SSI is typically associated with impaired wound healing, but the basis of these healing disturbances are still poorly understood. A typical feature of chronic wounds is the absence of the so-called granulation tissue, a highly vascularized tissue that is essential for the repair process (for a review, see ref. 4 ). Pathogens like S. aureus thus appear to have evolved efficient strategies to counteract the host’s inflammatory and angiogenic responses.

In this context, a group of cationic, noncovalently bound and partially secreted broad-spectrum adhesins of S. aureus has lately raised particular attention, as these proteins were found to have profound immunomodulatory properties (5 6 7 8) . Their significance as important virulence factors of S. aureus is supported by their ubiquitious distribution. In particular, expression of the extracellular adherence protein (Eap), also known as MHC class II analog protein (Map), was found in 97.9% of clinical isolates (9) . Moreover, eap transcription was recently reported to be mainly dependent on the regulator Sae, which has been connected with virulence gene expression during chronic S. aureus infections in humans and device-associated infections in mice (10 11 12) .

Previously, we demonstrated that binding of Eap to intercellular adhesion protein-1 (ICAM-1) severely impairs neutrophil recruitment in S. aureus-infected mice (7 , 8) . Because activated macrophages are the major source of angiogenic chemokines and growth factors, in particular, bFGF and vascular endothelial growth factor (VEGF) (13 , 14) , Eap’s interference with leukocyte recruitment will consequently have a negative effect on granulation tissue formation and healing, as was recently shown in an in vivo model of wound healing and angiogenesis in mice (15) . Currently, however, it is still unclear whether these effects of Eap are exclusively due to its anti-inflammatory activity or whether Eap also has a more direct impact on angiogenesis. It has been known for some time that staphylococcal surface proteins can bind basic growth factors like bFGF in a charge-dependent manner (16) . Moreover, it is becoming increasingly clear that endothelial responses to bFGF/VEGF are not solely mediated by their specific tyrosine kinase receptors but rather require the sequestration of a number of coreceptors together with FGF receptor 1 (FGFR-1) and VEGF receptor 2 (VEGFR-2/ KDR/Flk-1) (17 18 19 20 21 22) . Several endogenous inhibitors of angiogenesis, including endostatin, thrombospondin, and platelet factor 4, were previously reported to mediate their activities via interaction with some of these coreceptors, in particular, integrins and heparan sulfate proteoglycans (HSPG) (23 24 25 26 27) . These receptors and/or their ligands were also identified as possible targets of Eap and related proteins, which were shown to bind several extracellular matrix (ECM) proteins, including fibronectin and vitronectin, immunoglobulins like IgG and ICAM-1, and heparan sulfate (7 , 28 29 30) .

In this study we thus addressed the question, whether 1) Eap does interfere with angiogenic responses independent from its anti-inflammatory functions, 2) such an effect would be restricted to its interference with VEGF/bFGF interactions with their specific receptors, and 3) such an effect would be explainable by the induction of corresponding alterations in angiogenic signal transduction.

MATERIALS AND METHODS

Cell culture
Human umbilical vein endothelial cells (HUVEC) were isolated as described previously (31) . Bovine retinal endothelial cells (BREC) were kindly provided by Drs. S. Zink and P. Rosen (University of Düsseldorf, Germany) (32) . Cells were maintained on fibronectin-coated culture dishes in endothelial cell growth medium (PromoCell, Heidelberg, Germany) at 37°C under 5% CO2 in a humid atmosphere.

Eap purification
Eap was purified from Staphylococcus aureus strain Newman, as described previously (15) . In brief, cell wall proteins were obtained by lithium chloride extraction, and the extract subsequently adsorbed in batch onto SP Sepharose (Amersham-Pharmacia, Freiburg, Germany). After stepwise elution with increasing NaCl concentrations, the pooled eluted fractions (between 600 and 800 mM NaCl) were sterile filtered, and further purified by cation exchange chromatography on a Mono S 10/100 GL tricorn column and afterward by gel filtration using an Superdex 75 HR 10/30 column operated on the ÁKTA fast performance liquid chromatography system (Amersham-Pharmacia, Freiburg, Germany). Protein concentration and purity of the product were checked by SDS-PAGE and Bradford assay, respectively. Purified Eap was found to be free of detectable endotoxin.

Real-time polymerase chain reaction
HUVEC were serum-starved overnight in endothelial cell basal medium (PromoCell) with 0.5% FCS and then stimulated as indicated in fresh medium. After 12 h, total RNA was isolated using the Nucleospin RNAII kit from Machery-Nagel (Düren, Germany). Reverse transcription was performed with the high-capacity cDNA Archive kit from Applied Biosystems (Weiterstadt, Germany). 75 ng of cDNA per reaction were used in real-time polymerase chain reaction (PCR), performed on an ABI Prism 7000 Sequence Detection System instrument using the SYBR Green Master Mix kit from Applied Biosystems (Foster City, CA). Primers were designed with the Primer Express Software version 2.0.0. and amplicons chosen to span an intron region. Primer sequences for the decay- accelerating factor (DAF) were 5'-CATGATTGGAGAGCACTCTATTTATTG-3' forward and 5'-TTGGTGGGACCTTGGAAGTTAG-3' reverse, and for the "tissue factor" (TF) 5'-CCCCAGAGTTCACACCTTACCT-3' forward and 5'-CACTTTTGTTCCCACCTGTTCA-3' reverse. Data were analyzed according to the comparative Ct method and were normalized by ß-actin expression in each sample. Primer sequences for ß-actin were 5'-TGAGGCACTCTTCCAGCCTT-3' forward and 5'-CACTTCATGATGGAGTTGAAGGTAGT-3' reverse. Melting curves for each PCR reaction were generated to ensure the purity of the amplification product.

Endothelial cell proliferation. [3H]thymidine incorporation assay
HUVEC were seeded on fibronectin-coated, 24-well plates and grown to 60–70% confluence. Plates were washed twice with HBSS (Biochrom, Berlin, Germany), and cells were incubated in serum-reduced basal medium (endothelial cell basal medium with 28 mM HEPES, supplemented with 0.5% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin) for 18 h. Cells were then transferred to medium containing 0.1% FCS and 0.5% BSA and stimulated with the factors indicated for 24 h. During the last 4 h, 1 µCi [3H]thymidine was added per well. Cells were washed twice with ice-cold PBS, and DNA was acid-precipitated by incubation with 10% w/v TCA for 20 min at 4°C. Wells were washed once with ice-cold 10% w/v TCA, the contents solubilized in 0.2 M NaOH with 1% SDS, and wells were rinsed once with 0.2 M HCl. [3H]thymidine incorporation was determined by liquid scintillation counting in an Wallac 1410 instrument (Perkin Elmer Wallac, Freiburg, Germany).

Cell number counts
HUVEC were plated onto 96-well plates and incubated for 12 h, after which the medium was changed to MCDB-131 (CC-Pro, Neustadt, Germany) containing 0.05% FCS. Cells were then incubated for 72 h in the absence or presence of mitogens or Eap as indicated. Cells were subsequently collected by trypsinization, and the total cell number was determined with a Casy-Counter (Schärfe System, Reutlingen, Germany).

Sprouting assay
Sprouting assays were performed according to the method described by Nehls and Drenckhahn (33) . In brief, BREC were grown to confluence on Cytodex-3 microcarrier beads. Three hundred milliliters of a fibrinogen solution (1.8 mg/ml) with 200 U/ml aprotinin as protease inhibitor was placed together with 50–80 microcarrier beads per well into a 24-well culture plate. Polymerization was started by addition of 0.65 U/ml thrombin into each well, and plates were incubated for 20 min at room temperature. Medium was exchanged against endothelial cell basal medium with 0.5% FCS containing either 2 ng/ml bFGF or 0.9% saline as negative control. Eap was added at the concentrations indicated, and samples were assayed in triplicate. Plates were then incubated for 48 h and subsequently fixed in 3% w/v formaldehyde in PBS. Tube formation was assessed by counting all capillaries, which were at least as long as the bead diameter (average 150 µm) at 100 x magnification in a light microscope.

Western blot analysis
Cells were seeded in endothelial cell basal medium containing 0.5% FCS; they were transferred to basal medium with 0.5% BSA for 4 h on the following day, and then stimulated as indicated. Inhibitors or Eap were included at the concentrations and for the times stated. GF109203X and manumycin A were purchased from Calbiochem (San Diego, CA). Cells were washed twice with ice-cold PBS and lysed on ice by the addition of 200 µl of radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1% w/v IgepalTM, 0.25% w/v desoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM NaF, 0.5 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 2 µg/ml aprotinin, 5 µg/ml leupeptin, 10 µg/ml pepstatin A, and 0.2 mg/ml PMSF). Cell debris was removed by centrifugation at 10,000 g for 5 min at 4°C. Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, München, Germany), and 50 µg of total cellular protein was subjected to SDS-PAGE on 10% gels. Samples were electroblotted onto nitrocellulose membrane and regular transfer confirmed by staining with Ponceau S. Blots were blocked and incubated with primary antibodies corresponding to the recommendations of the supplier of the respective antibody (Ab): antiphosphoThr180/Tyr182-p38, antiphosphoSer473-Akt, and antiphosphoSer217/221-MEK1/2 rabbit polyclonal antibodies, and antiphosphoThr202/Tyr204-ERK1/2 mouse monoclonal were purchased from Cell Signaling Technology (Beverly, MA), anti-ERK1/2 rabbit polyclonal from Upstate Biotechnology (Lake Placid, NY), and anti-Raf-1 rabbit, anti-PKC{delta} goat polyclonal as well as anti-Egr-1 rabbit polyclonal from Santa-Cruz Biotechnology (Santa Cruz, CA). Immunoreactive bands were visualized on X-ray films using respective horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories, München, Germany) and enhanced chemiluminescence (ECL, Amersham-Pharmacia, Freiburg, Germany).

Preparation of cytosol and membrane fraction
HUVEC were treated, as described above, but scraped into ice-cold homogenization buffer (10 mM HEPES pH 7.4, 1 mM EDTA, 10 mM ß-glycerol phosphate, 0.1 mM DTT, 1 mM NaF, 0.5 mM sodium orthovanadate, 2 µg/ml aprotinin, 0.2 mg/ml PMSF, 5 µg/ml leupeptin, 10 µg/ml pepstatin A) and lysed by sonication (3 times, 5-s bursts, 50 W) on ice. Whole cells and nuclei were removed by centrifugation at 500 g for 2 min at 4°C. Membrane fraction was separated by centrifugation at 20,000 g for 30 min at 4°C. The supernatant containing the cytosolic fraction was preserved, and the pellet was dissolved in homogenization buffer containing 1% Triton X-100 by short sonication. Western blot analysis was performed as described above.

Pull-down assay
HUVEC were grown to 80–90% confluence and serum starved overnight prior to stimulation without or with VEGF165 (10 ng/ml, 5 min), bFGF (100 ng/ml, 15 min), in the absence or presence of Eap (10 µg/ml). Cells were washed with ice-cold PBS and lysed in MLB (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% w/v Nonidet P-40, 10 mM MgCl2, 1 mM EDTA, 25 mM NaF, 1 mM sodium orthovanadate, 10% w/v glycerol and protease inhibitors). Lysates were centrifuged for 5 min at 13,000 g and active Ras was precipitated with 10 µg RBD-conjugated agarose (Upstate Biotechnology, Lake Placid, NY) for 30 min at 4°C. Precipitates were washed thrice with lysis buffer, resuspended in 2 x sample buffer, and analyzed by SDS-PAGE on 15% gels. Ras was detected using respective rabbit polyclonal antibody from Cell Signaling Technology.

RESULTS

Eap inhibits the angiogenic response of primary endothelial cells
Real-time PCR was employed to study the transcriptional up-regulation of TF and DAF as one of the early steps during endothelial activation. In agreement with previous reports (34 , 35) , we found mRNA levels of DAF to be raised approximately twofold by either VEGF165 or bFGF in HUVEC. In contrast, expression of TF was increased approximately fivefold by VEGF165, whereas bFGF exerted only an insignificant effect on TF expression (Fig. 1 A). In comparison, HUVEC that were cotreated with 20 µg/ml of purified Eap, displayed a strongly attenuated response, the induction being reduced by about half.


Figure 1
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  		Figure 1. Eap interferes with angiogenic response of EC primary cultures. A) mRNA levels of DAF and TF were determined by real-time PCR in HUVEC stimulated with either 100 ng/ml VEGF or bFGF in the absence (open bars)or presence (solid bars) of 20 µg/ml Eap. Results are the means ± SD of 3 independent experiments and shown as a fold increase over basal expression (basal = 1, indicated by dashed line); *P < 0.05; **P < 0.01; ***P < 0.001. B) HUVEC were grown to subconfluence in 24-well plates, serum-starved overnight (0.5% FCS), and then stimulated with either VEGF (25 ng/ml), bFGF (25 ng/ml), or medium alone (0.1% FCS and 0.5% BSA) for 20 h in the absence (solid bars) or presence (open bars) of 20 µg/ml isolated Eap. Cells were pulse-labeled with tritium-thymidine for another 4 h and analyzed for thymidine incorporation. Data represent mean counts per minute ± SD with n = 5. *P < 0.05; **P < 0.01; ***P < 0.001. C) HUVEC were treated with VEGF or bFGF in serum-reduced medium (0.2% FCS), in the absence or presence of increasing concentrations of Eap as indicated. Proliferation is expressed as relative change in cell number after 72 h (basal cell number=1, indicated by dashed line). Data are mean ± SD, n = 3. D) Eap inhibits capillary sprout formation. Capillary-like sprout formation of BREC was analyzed without (open bars) or in the presence (filled bars) of either 2 ng/ml bFGF or 25 ng/ml VEGF and together with the indicated concentration of isolated Eap. Data represent mean ± SD of 30 beads analyzed in triplicate each.

The proliferation of endothelial cells is essential for new vessel formation, and both bFGF and VEGF have been described as potent mitogens for cultured endothelial monolayers. Proliferation in the presence or absence of Eap was assessed by measuring de novo DNA synthesis, as well as by cell number counts. As shown in Fig. 1B , [3H]thymidine incorporation was raised 4.0 ± 1.6 and 8.7 ± 2.7 fold in HUVEC by VEGF165 and bFGF, respectively. In the presence of 20 µg/ml Eap, this mitogenic response was completely abrogated (1.28±0.66 and 1.36±0.17). The antiproliferative activity of Eap was concentration dependent, with complete inhibition seen at 20 µg/ml (Fig. 1C ).

An important aspect of neovascularization is the capacity of endothelial cells to differentiate into a branched, vascular network. Primary endothelial cells can be coaxed to form capillary-like structures in vitro, when they are grown in a 3D fibrin matrix containing an angiogenic stimulus. Analyzing the bFGF- and VEGF-induced capillary-like sprout formation of BREC in the presence and absence of Eap, a strong inhibitory effect on sprouting was observed (Fig. 1D ).

Eap inhibits VEGF165 and bFGF-induced ERK phosphorylation but has variable effects on Akt phosphorylation
Changes in endothelial cell functions are mediated by induction of specific signaling cascades. We thus examined the phosphorylation of key kinases in the absence or presence of Eap. Lysates of HUVEC stimulated with 100 ng/ml of either VEGF165 or bFGF were immunoblotted with phosphospecific antibodies directed against the activated forms of ERK1/2 (phospho-Thr202/Tyr204), p38 (phospho-Thr180/Tyr182), and Akt/PKB (phospho-Ser-473). VEGF treatment induced a strong phosphorylation of ERK1 and 2, with a maximal response seen at 5 min (Fig. 2 A). There was also a fast, albeit weaker phosphorylation of p38, whereas Akt/PKB phosphorylation progressed more slowly. The maximal effect was not seen before 15 min of stimulation. By 60 min, the phosphorylation levels of all three kinases had dropped to or below basal levels. One hour preincubation with Eap led to a significant reduction in the level of the subsequent VEGF165- triggered ERK1/2 phosphorylation without altering the kinetics of the response. Under the same conditions, Eap did not affect the VEGF165-induced p38 and Akt/PKB phosphorylation. This specific suppression of the MAPK pathway through Eap was not restricted to the VEGF165 elicited response, since similar results were obtained in bFGF-stimulated HUVEC. Here, the maximal responsein ERK1/2 phosphorylation was seen after 15 min, and this was again significantly lowered by pretreatment with Eap (Fig. 2B ). In addition, there was an augmental effect of Eap on the bFGF-induced Akt/PKB phosphorylation: while bFGF on its own caused only a marginal increase in Akt/PKB phosphorylation at serine 473, in the presence of Eap this was clearly enhanced.


Figure 2
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  		Figure 2. Eap interferes with ERK1/2 activation. Serum-deprived HUVEC were left untreated or incubated with 100 µg/ml Eap for 1 h before stimulation with 100 ng/ml of either VEGF (A) or bFGF (B) for the times indicated. After cell lysis, 50 µg of total cell protein per lane were analyzed by SDS-PAGE on 10% gels and submitted to Western blot analysis with antibodies specific for the phosphorylated forms of either ERK1/2, p38, or Akt. HUVEC (C) or BREC (D) were incubated with increasing concentrations of Eap as indicated. Stimulation periods were chosen as 5 or 15 min, depending on time points of maximal activation seen in (A) and (B). E) Densitometric evaluation of ERK1/2 (left) and Akt (right) phosphorylation levels, taking phosphorylation in the absence of Eap as 100% (n=3; *P<0.05; **P<0.01; ***P<0.001).

As depicted in Fig. 2C , the observed effects of Eap are concentration-dependent. Fig. 2E shows the densitometric evaluation of a typical experiment, taking the level of ERK1/2-phosphorylation achieved in the absence of Eap as 100%. In good agreement with a recent report, which found Eap’s impact on leukocyte-endothelial interactions to be maximal at ~30 µg/ml (8) , inhibition with 20 µg/ml Eap reached between 80 and 90% of the maximal effect observed. Whereas basal phosphorylation states were reduced to nondetectable levels by incubation with Eap, the inhibitory effects on the responses stimulated with 100 ng/ml of either VEGF or bFGF were never complete. Intracellular signal transduction pathways for VEGF-induced activation of ERK1/2 were previously claimed to be heterogeneous and to depend on the origin of the EC (36) . Consequently, we studied the effect of Eap on signal transduction in BREC for comparison. As shown in Fig. 2D and E , the results obtained with BREC during VEGF stimulation are indistinguishable from those obtained with HUVEC at low concentrations of Eap; however, at higher concentrations, inhibition becomes almost complete. In contrast to HUVEC, BREC responded to bFGF stimulation with a strong activation of Akt, and this, too, was effectively inhibited through Eap.

To test whether these Eap-induced changes in signal transduction may be responsible for the disturbances in biological function described above, we examined the effect of Eap on the induction of the transcription factor Egr-1. De novo synthesis of Egr-1 was previously reported to be necessary for transcriptional activation of TF expression (31) . As shown in Fig. 3 , induction of Egr-1 was decreased by Eap to a similar degree as transcriptional up-regulation of TF.


Figure 3
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  		Figure 3. Induction of Egr-1 is inhibited by Eap. Egr-1 synthesis in HUVEC was induced by VEGF treatment for the times indicated. Western blot analysis was performed against Egr-1 in 50 µg of total cell protein.

Inhibition of the MAPK cascade occurs upstream of ERK1/2, and downstream of PLC{gamma}
To delineate the target of Eap in more detail, we studied the effects of combinations of Eap, the PKC inhibitor GF109203X, and the farnesyl transferase inhibitor manumycin A on the VEGF-induced MAPK cascade. As farnesylation of Ras is a prerequisite for its homodimerization and localization to the plasma membrane, preincubation of cells with manumycin A indirectly interferes with Ras activation. As shown in Fig. 4 A, GF109203X and Eap had a substantial inhibitory effect on ERK1/2 phosphorylation, with the combined effect resulting in the complete blockage of the phosphorylation response. In comparison, manumycin A treatment on its own had only a minor impact on ERK1/2 phosphorylation, whereas concomitant treatment with Eap resulted again in an additive effect. To further test whether the impact of Eap would be independent of receptor activation, we examined the effects of Eap, GF109203X, and manumycin A on activation of the MAPK pathway during stimulation with the phorbol ester TPA. As demonstrated in the right panel of Fig. 4A the results are very similar to those seen under VEGF stimulation. The MAPKK MEK1/2 are placed directly upstream of ERK1/2 and downstream of Raf-1 in the signal cascade. They are activated through Raf-1 by phosphorylation at Ser-217/221 (37) . Immunoblotting with a phospho-Ser-217/221 specific Ab showed Eap to have similar effects on MEK activation as on ERK (Fig. 4A ). The VEGF-induced MEK1/2 activation is unusual in that it relies on PLC{gamma}-dependent activation of novel PKC{delta} (38) . Activation of PKC requires its translocation to the plasma membrane and autophosphorylation. As shown in Fig. 4B , Eap had no impact on the membrane translocation or phosphorylation levels of PKC{delta}. Consequently, the inhibitory effect of Eap on MAPK cascade induction in EC has to occur downstream of PLC{gamma} and upstream of MEK1/2 phosphorylation, which is at the level of either Ras or Raf-1.


Figure 4
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  		Figure 4. Inhibition through Eap occurs downstream of PLC{gamma}. A) Comparison of the Eap effect with known inhibitors and activators of VEGF-induced MAPK signal cascade. Serum-deprived HUVEC were stimulated with VEGF (100 ng/ml; left) or TPA (100 nM; right) for 5 min. Where indicated, cells were pretreated with GF109203X (5 µM, 30 min), manumycin A (20 µM, 4 h) or Eap (40 µg/ml, 60 min). Control cells were pretreated with DMSO. MAPK pathway activation was analyzed by immunoblotting with antiphospho-ERK1/2 and antiphospho-MEK1/2 antibodies. Total ERK1/2 was determined to confirm equal loading. B) Membrane translocation and activation of PKC{delta} are not affected by Eap. HUVEC were stimulated with TPA (100 nM) for the times indicated and cytosolic and membrane fractions isolated. Activation of PKC{delta} is accompanied by electrophoretic mobility shift.

Eap exerts its effect at the level of Ras but not at the level of Raf-1
Raf-1 constitutes an important relay point within the MAPK cascade, integrating positive and negative inputs from upstream pathways (for a review, see Ref. 37 ). Activation of Raf-1 is accompanied by a characteristic shift in electrophoretic mobility due to an increased phosphorylation state (39) . As expected, stimulation with TPA induced the occurrence of an additional, retarded band in HUVEC (Fig. 5 A). The intensity of this band was again reduced on treatment with Eap. Pull-down assays were used to investigate Ras activation. As shown in Fig. 5B , there was a profound reduction in activated, GTP-bound Ras after VEGF, as well as bFGF stimulation in Eap-exposed HUVEC, indicating interference of Ras activation as the earliest target of Eap blockage of MAPK pathway activation.


Figure 5
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  		Figure 5. Eap blocks Ras activation. A) Eap interferes with Raf-1 activation. HUVEC were serum-starved and then stimulated with 100 nM TPA for the times indicated. Activation of Raf-1 was determined by phosphorylation-induced shift in electrophoretic mobility. B) Eap blocks Ras activation. Pull-down assays were performed on HUVEC stimulated in the presence or absence of Eap.

DISCUSSION

We reported previously that S. aureus Eap interfered with leukocyte accumulation at inflammatory locations in vivo, presumably by disturbing leukocyte-endothelium interactions, as demonstrated in vitro (7 , 8) . In addition, we recently showed that this versatile protein severely impairs wound healing and angiogenesis in mice and that this effect may go beyond its interference with the inflammatory response (15) . The aim of this study thus was to elucidate the basis of Eap’s potential impact on endothelial function. The data presented here demonstrate for the first time that Eap selectively inhibits Ras-dependent signaling pathways by an as yet unidentified mechanism and that this inhibition is associated with a corresponding attenuation of angiogenic responses in isolated primary endothelial cells.

Angiogenesis involves the concerted activity of many cell types. To evaluate the specific role of Eap-endothelial interactions in this context, we tested its effect on the behavior of isolated endothelial cells in vitro. Cotreatment with Eap was found to severely impair or completely abrogate essential angiogenic responses elicited by bFGF or VEGF165 in macrovascular as well as microvascular cells, that is, the induction of gene transcription, proliferation, and differentiation into tubular networks. At concentrations of 40 µg/ml, Eap blocked the transcriptional activation response by ~50 to 60%, whereas proliferation, as well as tubular morphogenesis, were completely abrogated at Eap concentrations of 20 µg/ml or less. A common feature distinguishing these last two responses from the short-term effect of gene up-regulation is their need for sustained and/or tightly regulated ERK and Ras signaling (20 , 40) . Similarly acting endogenous inhibitors of angiogenesis, like platelet factor 4 (PF4), 16-kDa human prolactin (16 K hPRL), thrombospondin (TSP), angiostatin, or endostatin, were previously shown to have an impact on the signal transduction network of endothelial cells, activating proapoptotic pathways and/or inhibiting MAPK signal transduction (41 42 43 44 45) . To elucidate the potent antiangiogenic activity of Eap in more detail, we investigated its effect on the activation of proangiogenic phosphorylation cascades.

Being the two major angiogenic growth factors in physiological, as well as pathological neovascularization, the intracellular signaling cascades linked to the main endothelial receptors for VEGF165 and bFGF, i.e., VEGF receptor 2/KDR/Flk-1 and FGF receptor 1, respectively, have been relatively well characterized (compare Fig. 6 ). In particular, was it possible to associate certain angiogenic responses with the activation of a specific pathway: so is the activation of p38 MAPK essential to actin rearrangements, permeability changes, and migration, ERK1/2 MAPK mediate transcriptional induction and cell proliferation, while Akt activation is responsible for cell survival by inhibiting proapoptotic pathways, apart from its contribution to eNOS activation (for a review, see Ref. 46 ). Examining the activation of these three key kinases in HUVEC, we found Eap to cause a significant impairment of the VEGF165- and bFGF-induced ERK1/2 phosphorylation, while, at the same time, Akt and p38 MAPK activation were either not affected or, in the case of bFGF stimulation, even augmented. With the high concentrations of stimulants employed here, the inhibitory effect on ERK1/2 phosphorylation was not complete: the maximal inhibition levels obtained were between 50 and 60%, with an IC50 of ~150 nM.


Figure 6
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  		Figure 6. Schematic illustration of bFGF- and VEGF- induced signal cascades and proposed model of the effect of Eap on endothelial function. Activation of FGF receptor 1 (FGFR-1) by bFGF leads to binding of adaptor protein FRS2, which, in turn, recruits Grb2 complexed to docking protein Gab1 and guanidine nucleotide exchange factor Sos. Sos-mediated activation of Ras results in induction of the Raf/MEK/ERK-cascade-promoting proliferation, gene expression, and differentiation, as well as in activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway, which enhances cell survival through inhibition of proapoptotic proteins (a). Prevention of Ras activation by externally added Eap will block both of these pathways, whereas an alternative Ras-independent pathway of PI3K/Akt activation is actually supported by Eap (b). In contrast, Akt/PI3K induction on autotyrosinephosphorylation of activated VEGF receptor may occur via direct interaction of the regulatory subunit of PI3K with the receptor and is not affected by Eap. Central to the induction of the Raf/MEK/ERK-pathway by VEGF is the generation of the second messenger sn-1,2-diacylglycerol by activated PLC{gamma}. Diacylglycerol (DAG) is a physiological activator of protein kinase C (PKC), which, in turn, will phosphorylate Raf-1 directly, but, in addition, activates sphingosine kinase, the subsequent increase in local membrane S1P levels leading to a decrease in Ras-growth-associated protein (GAP) activity. This, together with the induction of GRP3 promoted by DAG and PKC, will cause activation of Ras.

Some antiangiogenic factors have previously been shown to act, at least in part, by interfering with VEGF and bFGF binding to endothelial cells (47 , 48) . This possibility was excluded here by demonstrating interference of Eap with activation of the Raf/MEK/ERK cascade on TPA exposure. Phorbol esters such as TPA mimic the function of sn-1,2-diacylglycerol by inducing the membrane translocation of PKC and the guanidine nucleotide exchange factor RasGRP3 and are able to induce the proliferation and tube formation of cultured endothelial cells in a tyrosine kinase receptor-independent manner (49 50 51) . The fact that the Eap-induced blockage of ERK1/2 activation does indeed occur downstream of PKC, was confirmed by the demonstrating that in the presence of Eap membrane translocation and activation of PKC{delta} was unaffected.

In the past, there has been some controversy over the relative contributions made by Ras-dependent vs. Ras-independent pathways during VEGF-triggered MAPK pathway activation (52 , 53) . Yashima et al., studying the VEGF-induced ERK1/2 activation in different endothelial cell types, came to the conclusion that in microvascular EC, the Ras-dependent branch is used preferentially, whereas PKC plays the dominant role in macrovascular EC (36) . Because the Eap blockage on stimulated ERK1/2 phosphorylation was incomplete in HUVEC, we investigated its effect on primary endothelial cells derived from a microvasuclar source, that is, BREC. Indeed, in these cells, a nearly complete inhibition of the MAPK pathway could be achieved at high Eap concentrations.

Within the MAPK cascade, Raf-1 has a special role in the integration of positive and negative crosstalk from other pathways (reviewed in Ref. 37 ). Raf-1 activation was previously described to be accompanied by a characteristic electric mobility shift, which was slightly decreased in the presence of Eap. Raf-1 activation is dependent on the small GTPase Ras, and Ras-GTP formation was finally identified as the first event in the MAPK pathway to be repressed by Eap.

In contrast to the Raf/MEK/ERK module, there is only very little known about the signal transduction components linking Akt activation to receptor autophosphorylation in EC. The effect of Eap on Akt serine 473 phosphorylation was found to depend on the particular cell type examined and the stimulant used. Because PI3-kinase induction upstream of Akt activation may occur in a Ras-dependent or -independent manner, the Eap-induced blockage of Akt phosphorylation observed in bFGF-stimulated BREC seem to indicate the involvement of Ras in these cells. In support of this assumption, Zubilewicz et al. previously demonstrated bFGF-induced Ras-dependent PI3-kinase activation in bovine chorocapillary EC (54) . Very much in contrast to these findings, bFGF alone was only a weak inductor of Akt in HUVEC, but this minor activation was increased ~2.5-fold by concomitant treatment with Eap.

Taken together, Eap thus does not only exert angiostatic activities comparable to those of established endogenous angiostatins, but there are also striking parallels in the impact on the endothelial signal transduction network bringing about these alterations. So was 16 K hPRL reported to affect VEGF signaling via inhibition of Ras activation, and PF4 was shown to inhibit VEGF-mediated ERK1/2 activation without affecting PI3-kinase induction (41 , 42) .

Clearly, elucidation of Eap activities at the membrane level may also shed some light onto signaling in eukaryotic cells. From the bacterial viewpoint, Eap appears to be the ideal adhesin: mediating staphylococcal binding to the ECM, as well as to the eukaryotic surface, while at the same time soothing the host’s response. The potent anti-inflammatory and antiangiogenic activities of Eap make this versatile protein a good candidate for future pharmacological application, for instance, in inhibiting pathological neovascularization as occurs in tumor growth, or curbing the overshooting host responses in sepsis.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Claudia Rubie for technical advice with qRT-polymerase chain reaction and Dr. Banhu Sinha for helpful discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft to M. Herrmann (H3 1850/6–1), K. T. Preissner (PR327/18–2), and T. Chavakis (CH279/2–1 and CH279/2–2). M. Hussain was supported by SFB492.

FOOTNOTES

2 Present address: Experimental Immunology Branch, NCI, Bethesda, MD, USA.

Received for publication March 28, 2006. Accepted for publication July 24, 2006.

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